Heparan sulfate (HS) and heparan sulfate proteoglycan (HSPG) play an important role in diverse biological systems, such as, i.e., proliferation, differentiation, homeostasis and viral pathogenesis (Perrimon and Bernfeld (2000) Nature 404: 725-728; Rosenberg et al. (1997) J. Clin. Invest. 99: 2062-2070; Shukla and Spear (2001) J. Clin. Invest. 108: 503-510). HSPG is present in almost every cell type in soluble form, as a component of the extracellular matrix (ECM), associated with plasma membranes, or segregated into intracellular granules (Lindahl et al. (1994) Thromb. Res. 75: 1-32). HSPGs modulate the biological activity of heparin-binding growth factors and cytokines through different mechanisms. For example, the optional binding of the growth factor to soluble, ECM-associated or cell-surface HSPGs may result in a fine control of the bioavailability of the protein. This is the case for TGF-β that binds betaglycan, a cell-associated PG (Massagué (1992) Cell 69: 1067-1070), and decorin (Yamaguchi et al. (1990) Nature (Lond.) 346: 281-284), which is present in the ECM, and for FGF-2 that binds basement membrane perlecan as well as cell-membrane syndecans (Avezier et al. (1994) Cell 79: 1005-1013; Samivirta et al. (1992) J. Biol. Chem. 267: 17606-17610). In addition, the association with HSPGs may stabilize the growth factor and protects it from proteolytic degradation (Saksela et al. (1988) J. Cell Biol. 107: 743-751; Sommer and Rifkin (1989) J. Cell. Physiol. 138: 215-220). Further, HSPGs modulate the access of growth factors to specific signaling receptors by different mechanisms (Ruoslahti and Yamaguchi (1991) Cell 64: 867-869). HSPGs can also control the intracellular fate of a growth factor (Rusnati et al. (1993) J. Cell. Physiol. 154: 152-161). In addition, transmembrane HSPGs themselves may transduce an intracellular signal, as suggested by the presence of highly conserved tyrosine residues in the C-terminal of all the members of the syndecan family, one of them fitting a consensus sequence for tyrosine phosphorylation. HSPGs may also activate an intracellular transduction signal by interacting directly with growth factor receptors (Revis-Gupta et al. (1991) Proc. Natl. Acad. Sci. U.S.A. 88: 5954-5958; Gao and Goldfarb (1995) EMBO J. 14: 2183-2190). (Special project Angiogenesis website http://www.med.unibs.it/˜airc/).
Whatever the mechanism(s) of regulation of growth factor activity by HSPGs, the binding of the same growth factor to different HSPGs may have different biological consequences. This is the case for syndecan (Ausprunk et al. (1981) Am. J. Pathol. 103: 353-366; Chemousov and Carey (1993) J. Biol. Chem. 268: 16810-16814), betaglycan (Andres et al. (1992) J. Biol. Chem. 267: 5927-5930) and perlecan (Avezier et al. (1994) Cell 79: 1005-1013), which are all able to bind FGF-2 but with different effects. Syndecan inhibits the mitogenic activity of FGF-2 (Mali et al. (1993) J. Biol. Chem. 268: 24215-24222) while perlecan promotes FGF-2-induced cell proliferation and angiogenesis (Avezier et al. (1994) supra). Conversely, modifications of HSPG composition can regulate the sensitivity of the cell to different growth factors. This may be of particular relevance when the spatial and temporal control of the activity of different growth factors must be tightly enforced. This possibility is exemplified by the shift in cell-surface HSPG properties from an FGF-2- to an FGF-1-binding phenotype in murine neuronal cells during embryonic development (Nurcombe et al. (1995) Science 260: 103-106). (Special project Angiogenesis website http://www.med.unibs.it/˜airc/).
IIS is an unbranched polysaccharide polymer covalently attached to the core protein of proteoglycans. The myriad of functions attributed to HS is a consequence of its ability to interact with proteins and to affect their stability, conformation, concentration, and activity (Esko and Lindahl (2001) J. Clin. Invest. 108:169-173; Turnbull (1999) in Cell Surface Proteoglycans in Signalling and Development, Vol. VI (eds. Lander, A., Nakato, H., Selleck, S., Turnbull, J. & Coath, C.) p. 13-21). The binding interactions of HS with proteins regulate a number of cellular functions. For example, HS is critical for the entry into cells of herpes simplex virus type 1 (Shukla et al. (1999) Cell 99: 13-22); the HS chain of syndecan-3 is found to play a significant role in modulating feeding behavior in mice (Reizes et al. (2001) Cell 106: 105-116); and HS modulates the association between fibroblast growth factors (FGFs) and their receptors (FGFRs) (Spivak-Kroizman et al. (1994) Cell 79: 1015-1024; Pellegrini et al. (2000) Nature 407: 1029-1034; Schlessinger et al. (2000) Mol. Cell. 6: 743-750).
The biological significance of HS is also manifested at the whole animal level, where genes involved in HS biosynthesis are deficient (Forsberg and Kjellen (2001) J. Clin. Invest. 108: 175-180). For example, mice lacking glucosaminyl N-deacetylase/N-sulfotransferase 1 (NDST1) and HS 2-O sulfotransferase (2-OST), enzymes required for modifications of HS, die neonatally (Fan et al. (2000) FEBS Lett. 467: 7-11; Bullock et al. (1998) Genes Dev. 12: 1894-1906); a homozygous mutation of one of the HS polymerases, EXT1, in mice leads to embryonic lethality due to a failure to gastrulate (Lin et al. (2000) Dev. Biol. 224: 299-311); Drosophila with mutations of UDP-D-glucose dehydrogenase or NDST lack wingless activity as well as FGF and hedgehog signaling pathways (Bellaiche and Perrimon (1998) Nature 394: 85-88; Binari et al. (1997) Development 124: 2623-2632; Lin and Perrimon (1999) Nature 400: 281-284); and altered expression of HS proteoglycan leads to various human diseases, such as chondrodystrophic myotonia (Arikawa-Hirasawa et al. (2001) Nat. Genet. 27: 431-434) and hereditary bone disorders (McCormick et al. (1998) Nat. Genet. 19: 158-161).
Other oligosaccharides have a similar structure and dependence upon functional groups for binding to proteins. These include other GAGs, such as heparin and hyaluronic acid (HA); galactosaminoglycans, such as e.g., chondroitin-4-sulfate (C4S), chondroitin-6-sulfate (C6S), and dermatan sulfate (DS); and sulfated polylactosamines, such as, e.g., keratan sulfate (KS). Heparin, which is found in mast cells, has a similar structure to the sulfated regions of IIS. Chondroitin sulfates play a major role in the growth and repair of cartilage and consist of high viscosity mucopolysaccharides that act as the flexible connecting matrix between the protein filaments in cartilage to form a polymetric system. Chondroitin sulfates have been used in conjunction with glucosamine to treat osteoarthritis. A classification of some proteoglycans on the basis of their localization, core protein, and oligosaccharide content is provide in Table 1.
TABLE 1Classification of proteoglycans on the basis of their localizationand type of core proteinMr of thecore proteinLocalizationGAG-chain(kD)Principal membersECMHA, CS, KS225-250aggrecan, versicanCollagen-CS, DS, KS 40decorin, biglycanassociatedfibromodulinBasementHS120perlecanmembraneCell-surfaceHS, CS33[a]-60[b]-92[c]syndecans[a],glypican[b],betaglycan[c],CD44E, cerebroglycanIntracellularheparin, CS17-19SerglycingranulesCS, chondroitin sulfate;DS, dermatan sulfate;KS, keratan sulfate;HA, hyaluronic acid;HS, heparan sulfate.http://www.med.unibs.it/~airc/hspgs.html (Sep. 26, 2002).
Unlike DNA and proteins, in which sequence diversity is generated by nucleic acid and amino acid sequence variations, the sequence diversity of oligosaccharides such as HS is generated by heterogenous enzymatic modifications in the Golgi apparatus. HS is initially synthesized in the Golgi apparatus as non-sulfated copolymers attached to HS proteoglycan core proteins by sequential addition of D-glucuronic acid (G1cA) alternating with N-acetyl D-glucosamine (G1cNAc) catalyzed by HS polymerases. The oligosaccharide chain then undergoes various modification steps, which include N-deacetylation and N-sulfation of glucosamine, epimerization of GIcA to L-iduronic acid (IdoA), 2-O sulfation of uronic acid and 6-O sulfation and 3-O sulfation of glucosamine. All of the modification steps are catalyzed by different enzymes and the process is selective in terms of the position and the number of modifications in a chain, leading to extensive sequence diversity (Rosenberg et al. (1997) supra; Esko and Lindahl (2001) supra).
The heterogeneity of HS modifications may be due, for example, to differential expression or activity of these enzymes. With the exception of single isoforms for 2-O sulfotransferase (Kobayashi et al. (1997) J. Biol. Chem. 272: 13980-13985) and epimerase (Li et al. (1997) J. Biol. Chem. 272: 28158-28163), five isoforms have been cloned for 3-O sulfotransferase (3-OST) (Shworak et al (1999) J. Biol. Chem. 274: 5170-5184), four isoforms have been cloned for 6-O sulfotransferase (6-OST) (Habuchi et al. (1998) J. Biol. Chem. 273: 9208-9213) and four isoforms have been cloned for N-deacetylase/N-sulfotransferases (NDST) (Eriksson et al. (1994) J. Biol. Chem. 269: 10438-10443). Each isoform has its own substrate specificity, tissue expression pattern and, thus, unique function (Rosenberg et al. (1997) supra; Esko and Lindahl (2001) supra). For example, 3-OST-1 is specifically responsible for sulfating the antithrombin-III (AT-III) binding sequence and 3-OST-3A is critical for gD binding of herpes simplex virus (Shukla et al. (1999) supra). Tissue-specific and developmentally regulated expression of these isoforms produces HS chains with distinct sequences, enabling HS chains to interact with a broad array of protein ligands that modulate a wide range of biological functions involved in development, differentiation, homeostasis, and bacterial/viral entry.
Many proteins bind to HS (i.e., HS-binding proteins), including the proteins of the circulatory system, growth factors, receptors, adhesion proteins, enzymes, cytokines, chemokines, protease inhibitors and virus proteins. However, the oligosaccharide features (e.g., functional groups and their modification enzymes) required for binding to HS-binding proteins been defined for only a few proteins (Esko and Lindahl (2001) supra; Lindahl et al. (1998) J. Biol. Chem. 273: 24979-24982). For example, the way in which various HS proteoglycans (HSPG) discriminate among binding partners, e.g., heparin-binding growth factors, depends upon their different core proteins, the high heterogeneity of oligosaccharide composition, and on the possibility that both the protein moiety and oligosaccharide-chains may interact with different binding partners. For instance, betaglycan, an HSPG, can exist as a naked core protein and the presence and composition of the oligosacharide chains of betaglycan can be regulated in response to FGF-2 (Lopez-Casillas et al. (1991) Cell 67: 785-795). FGF-2 itself binds the oligosaccharide-chain of betaglycan while the core protein can interact with TGF-β (Andres et al. (1992) J. Biol. Chem. 267: 5927-5930). Also, the number and fine structure of HS chains in syndecan 1 vary in different tissues and in relation to cell differentiation (Bernfield and Sanderson (1990) Phil. Trans. R. Soc. Lond. 327: 171-186). Thus, different sulfated groups and distinct oligosaccharide sequences of the oligosaccharide chain are responsible for the binding to different growth factors (Special Project Angiogenesis website, http://www.med.unibs.it/˜airc/, Sep. 26, 2002). Thus, HSPGs are characterized by a structural variability that appears to be highly regulated and that offers numerous possibilities for selective interactions with different growth factors, cytokines, and chemokines.
FGF and FGFR act on a wide spectrum of tissues and cell types and play critical roles in various biological processes, such as cell proliferation, differentiation, migration, embryonic development, tissue maintenance, angiogenesis, tumor growth and wound healing (Burgess and Maciag (1989) Ann. Rev. Biochem. 58: 575-606; Galzie et al. (1997) Biochem. Cell. Biol. 75: 669-685; Givol and Yayon (1992) FASEB J. 6: 3362-3369; Martin (1998) Genes Dev. 12: 1571-1586). Currently, there are 23 known FGFs and 5 types of FGFRs (Sleeman et al. (2001) Gene 271: 171-182). FGF1 and FGF2 were first to be isolated and were called acidic and basic FGF, respectively. Studies performed primarily on FGF2, have identified a stretch of basic residues in the polypeptide chain as participating in the heparin-binding site (Faham et al. (1996) Science 271: 1116-11120). FGFRs are members of the receptor tyrosine kinase superfamily. These receptors contain an intracellular tyrosine kinase domain, a trans-membrane region and an extracellular region containing three immunoglobulin (Ig)-like domains (Givol and Yayon (1992) supra). All FGFRs contain a heparin/HS binding site consisting of a stretch of 18 conserved residues in the second Ig-loop (Kan et al. (1993) Science 259: 1918-21). The amino-terminal Ig-like domain I is dispensable and receptor variants containing only the Ig-like domain II and III exhibit an equivalent degree of binding to FGFs as the variants containing all three domains (Givol and Yayon (1992) supra). All receptors show redundant specificity for ligand binding, where one receptor may bind to several FGFs and one FGF may bind to several receptors. FGF1 interacts with all the four known FGFRs and their isoforms. (Special project Angiogenesis website http://www.med.unibs.it/˜airc/).
Direct involvement of heparan sulfate or heparin in the molecular association between FGF and its receptor is essential for FGF activated signal transduction (David (1993) FASEB J. 7: 1023-30; Kan et al. (1993) supra; Lundin et al. (2000) J. Biol. Chem. 275: 24653-24660; Spivak-Kroizman et al. (1994) Cell 79: 1015-1024). Heparan sulfate is required for the biological activity of FGF during Drosophila development (Lin et al. (1999) Development 126: 3715-3723). At normal concentrations, FGFs do not induce cell growth in HS deficient cells (Ornitz et al. (1992) Mol. Cell. Biol. 12: 240-247), but this induction can be restored by the addition of exogenous heparin (Rapraeger et al. (1991) Science 252: 1705-1708). The identification of HS as an active and essential component of the FGF:FGFR:HS signaling complex suggests that FGF activity and specificity may be modulated by HS and in turn, by enzymes that synthesize and degrade HS. It is of great pharmaceutical interest to design small oligosaccharides capable of modulating FGF signaling.
Although the importance of HS in FGF signaling is well documented, the exact structural roles of IIS in the signaling complex are less well characterized. One key issue concerns the minimum size of HS required for FGF signaling. The size of HS reflects the spatial arrangement of FGF and FGFR and thus is critical for establishing a model for FGF signaling complex. So far, various signaling complex models have been proposed based on crystallographic studies, and, in each case, a different optimal length of HS was postulated (DiGabriele et al. (1998) Nature 393: 812-817; Pellegrini et al. (2000) supra; Plotnikov et al. (1999) Cell 98: 641-650; Schlessinger et al. (2000) Mol. Cell. 6: 743-750). Octasaccharide was proposed to be the minimal HS in some models, because shorter oligosaccharides would be incapable of connecting both ligand and receptor (Pellegrini et al. (2000) supra; Plotnikov et al. (1999) supra). In another case, heparin hexasaccharide was proposed to be sufficient to promote receptor dimerization (Schlessinger et al. (2000) supra). The shortest biologically active heparin oligosaccharide has been determined to be octasaccharide (Ornitz et al. (1992) supra), hexasaccharide (Gambarini et al. (1993) Mol. Cell. Biochem. 124: 121-129; Zhou et al. (1997) Eur. J. Cell. Biol. 73: 71-80), trisaccharide, and even disaccharide (Ornitz et al. (1995) Science 268: 432-436; Ostrovsky et al. (2002) J. Biol. Chem. 277: 2444-2453). These contradictory findings necessitate a more accurate method to determine the size of HS in FGF signaling complexes.
The stoichiometry of HS in the FGF molecular signaling complex has also been a subject of controversy. Intracellular signaling is believed to be initiated by receptor dimerization and trans-phosphorylation (Bellot et al. (1991) EMBO J. 10: 2849-2854; Heldin (1995) Cell 80: 213-223). In broad terms, three distinct models, with the ability for FGFR dimerization but different stoichiometry for FGF and ITS have been proposed based on biochemical and crystallographical evidence (Delehedde et al. (2002) Biochem. J. 9: 366:235-244). In the first model, an FGF dimerizes two FGFRs, with a single HS chain binding both FGF and its receptor (Hsu (1999) Biochemistry 38: 2523-2534; Springer et al. (1994) J. Biol. Chem. 269: 26879-26884). In the second model, a single HS chain binds two FGFs, which in turn bind two FGFRs (Kwan et al. (2001) J. Biol. Chem. 276: 23421-23429; Pellegrini et al. (2000) supra). In the third model, one each of FGF, HS and FGFR first form an FGF:HS:FGFR complex, two of which then in turn dimerize (Schlessinger et al. (2000) supra).
Biological studies show that HSs from different tissues or developmental stages have different fine structures (Allen et al. (2001) J. Cell. Biol. 155: 845-858; Lindahl et al. (1998) supra) and can activate or even inhibit FGF signaling pathways (Pye et al. (2000) Glycobiology 10: 1183-1192; Zhang et al. (2001c) J. Biol. Chem. 276: 41921-41929). It is believed that this phenomenon is caused by switching of critical functional groups on the HSs (Lindahl et al. (1998) supra; Pye et al. (2000) supra). The critical functional groups on HS that interact with FGFs or FGFRs have been investigated previously. (Wu et al. (2002) supra; Guimond et al. (1993) J. Biol. Chem. 268: 23906-23914; Lyon and Gallagher (1998) Matrix Biol. 17: 485-493; Maccarana et al. (1993) J. Biol. Chem. 268: 23898-23905; Pye et al. (2000) supra; Turnbull et al. (1992) J. Biol. Chem. 267: 10337-10341; Zhou et al. (1997) supra) (Loo et al. (2001) J. Biol. Chem. 276: 16868-16876; McKeehan et al. (1999) J. Biol. Chem. 274: 21511-21514; Ostrovsky et al. (2002) supra). For example, 2-O sulfation at an iduronic acid are critical for FGF2 binding (Maccarana et al. (1993) supra) and 6-O-sulfate groups are important for FGFR4 binding (Loo et al. (2001) supra), but no information is available about the critical groups on HS mediating binding to FGF:FGFR binary complex. These critical groups may be different from those binding to individual FGFs and FGFRs, because the binding environment in the FGF:FGFR complex is different from that in individual FGFs or FGFRs (Plotnikov et al. (1999) supra). The study of these critical functional groups are important, because HS binding to the FGF:FGFR binary complex directly affects the formation of the FGF:HS:FGFR ternary complex, which is a prerequisite for the activation of FGF receptors.
There is little doubt that identifying the functional features on oligosaccharides will help to settle fundamental questions regarding development, physiology and the behavior process. However, neither molecular cloning nor sequencing technology are sufficient methods for obtaining large volumes of heterogenously modified oligosaccharides such as HS (Venkataraman et al. (1999) Science 286: 537-542). Such oligosaccharides may be heterogeneous in terms of sequence and size, and their binding partners may recognize motif structures rather than single, defined sequences. Most studies of heterogenously modified oligosaccharides require affinity purification to obtain a homogeneous population. However, the interaction between a heterogenously modified oligosaccharide and its binding partner(s) may be relatively weak and, because the source of heterogenously modified oligosaccharides is usually limited, it is almost impossible to obtain a homogeneous oligosaccharide sample by affinity purification. In addition, most current methods for studying oligosaccharide-protein interaction involve immobilization or chemical labeling of either component, which may introduce more difficulties and artifacts. Thus, a need exists for methods that can rapidly and accurately characterize the structural and/or functional features of a heterogenously modified oligosaccharide, such as HS, as well as to study oligosaccharide-binding partner interactions and to identify agents capable of interfering with, or enhancing, oligosaccharide-binding partner interactions and/or activity.